Rabbit muscle phosphorylase derivatives with oligosaccharides covalently bound to the glycogen storage site.
Linear maltooligosaccharides, e.g., maltoheptaose or terminal 4-O-methylmaltoheptaose, activated by cyanogen bromide, react covalently with rabbit muscle phosphorylases b and a (EC 2.4.1.1). Site-specific modification prevents further binding to glycogen and shifts the phosphorylase a tetramer-dimer equilibrium in favor of the dimer. Use was made of these properties to separate by affinity chromatography and gel filtration phosphorylase a dimers with specifically bound oligosaccharide from unspecifically modified products. The phosphorylase a-maltoheptaose derivative carries one oligosaccharide residue per monomer and can be distinguished from the native enzyme by its electrophoretic mobility in polyacrylamide gels or by affinity electrophoresis. Phosphorylase a preparations with covalently bound maltooligosaccharides are enzymatically active in the presence of a primer and alpha-D-glucopyranose 1-phosphate (glucose-1-P). Methylation of the nonreducing chain terminus of the bound oligosaccharide has no effect on glycogen synthesis. These findings exclude the participation of bound oligosaccharides in chain elongation. Purified covalent phosphorylase a-maltoheptaose complexes are stable dimers. They are no longer activated by glycogen. The properties of covalently modified phosphorylase-oligosaccharides are consistent with and provide direct evidence for the existence of a glycogen storage site in rabbit muscle phosphorylases. Covalent occupation of the storage site renders the affinity of glucose-1-P to phosphorylase a independent of modulation by glycogen, supporting the assumption that the glycogen storage site is involved in interactions with the catalytic site.
D000818:Animals; D001665:Binding Sites; D003460:Crystallization; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D009844:Oligosaccharides / Q000494:pharmacology*; D010762:Phosphorylase a / Q000302:isolation & purification / Q000378:metabolism; D010763:Phosphorylase b / Q000302:isolation & purification / Q000378:metabolism; D006005:Phosphorylases / Q000378:metabolism*; D011817:Rabbits
D009844:Oligosaccharides; C021705:maltooligosaccharides; D010762:Phosphorylase a; D010763:Phosphorylase b; D006005:Phosphorylases